Vanderbilt University

NIH Research Projects · FY 2026 · 2020-07

Project Summary Lowered functional β-cell mass increases the risk of type 2 diabetes (T2D). β-cell production begins in early embryonic neogenesis, when endocrine progenitor cells expressing the transcription factor Ngn3 differentiate into pro-β cells. These cells then proliferate and mature during early postnatal life to establish a functional β-cell mass. In adults, this mass further increases in response to obesity-related insulin resistance via increased proliferation and function (i.e., adaptation); it can also decrease due to β-cell failure when metabolic stress becomes too severe. How genetic and environmental factors work together to regulate this mass is not well understood, which prevents effective T2D intervention by inducing functional β-cell mass. This gap is further complicated by β-cell heterogeneity, with β cells in each individual consisting of subtypes with distinct fitness to proliferate, survive, and function. A pressing need is to study the proportional regulation of β-cell subtypes and their respective fitness, which is particularly challenging due to the lack of methods to label specific β-cell subtypes. We have reported that the mouse islet progenitors consist of two subtypes, the Myt1⁺Ngn3⁺ (M+N+) and Myt1⁻Ngn3⁺ (M-N+) cells. Myt1 is a transcription factor that is expressed in all islet cells but for reasons still unknown is unequally expressed in Ngn3+ cells. The M+N+ and M-N+ progenitor subtypes have different DNA methylation (DNAme) patterns and transcriptomes. Using our split-Cre-based lineage tracing, we showed that the M⁺N⁺ progenitors gave rise to β-cell subtype (M+N+βCs) of better fitness than those of M-N⁺ progenitors (M- N+βCs). These β-cell subtypes have distinct transcriptomes and DNAme patterns, with the former expressing higher levels of Dnmt3a. Dnmt3a is required for the creation and maintenance of DNAme patterns. It promotes β-cell function by repressing a few disallowed genes in β cells, suggesting its unequal expression could induce and preserve the differential DNA methylation patterns in the two β-cell subtypes. Underscoring the significance of our findings, β-cell subtypes similar to the mouse M+N+βCs and M-N+βCs can be identified in human islets, with the proportion of high-fitness M+N+βCs reduced in T2D donors. In addition, we found that maternal obesity, a condition that happens in over 30% of all human pregnancies and is a well-established T2D risk factor in human offspring, reduces the proportion of the high-fitness β-cell subtype and predisposes offspring β cells to poor fitness, phenotypes that coincide with reduced expression of Dnmt3a in EPCs. These findings suggest that genetic and maternal obesity signals converge on the DNAme patterns of islet progenitors to predispose the future functional β-cell mass and risk of T2D by aberrant β-cell subtype allocation. In this application, we will first examine how the DNAme patterns are induced and preserved in the β-cell subtypes in mouse and human islets (Aim 1). We will then test how maternal obesity regulates the allocation of fetal β-cell subtypes and predisposes to poor functional β-cell mass. We expect to identify maternal nutrition-related factors and pathways that can be targeted to reduce the risk of T2D in human subjects.

NIH Research Projects · FY 2026 · 2020-06

ABSTRACT Ribosomes carry out protein synthesis in all cells, interpreting the information contained in the mRNA to produce the proper amount of the correct protein. In addition, ribosomes also mediate mRNA quality control. Thus, misassembled, or damaged ribosomes as well as insufficient ribosome numbers can affect the sequence and abundance of proteins and mRNAs, thereby disrupting protein homeostasis. This can lead to a number of diseases, demonstrating the importance of ensuring ribosomes are accurately assembled, and produced in the correct numbers. Using a combination of biochemical, genetic, genomic, and structural tools, we will (i) investigate mechanisms that enable ribosome production by avoiding RNA folding traps and dead-end intermediates, (ii) dissect quality control pathways to identify misassembled intermediates, (iii) study how defective ribosomes are identified for repair or degradation, and (iv) explore how ribosomes are remodeled to alter translation. In the first part, we will build on work in the current funding period to study the role of ordered helix formation and snoRNA binding for rRNA folding. This work has implications for the larger field of RNA folding, and RNP assembly, as well as the ribosome heterogeneity community, which is focused on snoRNA-mediated modifications. Our work indicates that snoRNA deletions affect ribosome assembly, which must be broadly considered in the study of ribosome heterogeneity. In the second component, we will extend our work on quality control to investigate a possible proofreading mechanism that quality checks the formation of the key functional feature of the small ribosomal subunit, the decoding site. In the third area, we will study the mechanisms of ribosome repair and degradation to decipher how cells make decisions about different outcomes from one complex, collided disomes. In the last section, we will study how the ubiquitin proteasome system is linked to ribosome remodeling via degradation of released RPs, and then dissect additional biological roles for these remodeled ribosomes. Together, the proposed work will provide a comprehensive picture of ribosome homeostasis, which underpins protein homeostasis and is thus critically important to prevent human diseases including cancer and neurodegeneration.

NIH Research Projects · FY 2025 · 2020-06

PROJECT SUMMARY DNA replication in eukaryotes is carried out multiprotein complexes that carry out a variety of enzymatic activities required for faithful genome duplication. Regulation and coordination of these activities at replication forks are essential for genomic stability. The replisome regularly encounters impediments that stall the replication fork, including DNA damage, aberrant DNA structures, and transcription conflicts. Stalled forks are a major source of genomic instability and underlie diseases including cancer. Replication-repair pathways serve to stabilize, repair, and restart stalled forks. However, the molecular mechanisms of these pathways are poorly understood. We are addressing this gap in knowledge by determining structures and mapping interactions within and among multiprotein-DNA complexes and combining this information with their biochemical and cellular functions. Our long-term goal is to decipher the molecular mechanisms and regulation of replication-repair pathways to understand how errors in these processes lead to disease. We are currently focused on three enzymatic activities essential for faithful genome duplication—(1) initiation of DNA synthesis by DNA polymerase (pol) α–primase, (2) fork reversal and template switching by ATP-dependent DNA translocases, and (3) DNA glycosylase mediated repair of interstrand DNA crosslinks (ICLs). (1) Polα–primase is a core component of the eukaryotic replisome that initiates DNA synthesis by generating chimeric RNA-DNA primers necessary for replicative synthesis by polδ and polε. Despite the importance of this critical activity, how polα–primase primes synthesis in the context of the replisome and how the primer is transferred to polδ for Okazaki fragment synthesis are unknown. We are determining structures of polα–primase with nucleic acid substrates and protein partners at various stages of its catalytic cycle and visualizing conformational states by electron microscopy and biophysical approaches. (2) Fork reversal involves remodeling a stalled fork into four-way junctions to prevent fork collapse and facilitate replication restart. Our work will address how HLTF, SMARCAL1, and FBH1 provide unique repair activities at damaged forks, their mechanisms of fork reversal, and how the ubiquitin ligase activities of HLTF and FBH1 regulate fork reversal. (3) Abasic (AP) sites are one of the most abundant forms of DNA damage and react with the opposite strand to form an AP-ICL. The NEIL3 glycosylase unhooks AP-ICLs at convergent replication forks by an unknown mechanism. Our previous work defined NEIL3’s specificity for AP-ICLs in a particular arrangement and spacing from a fork junction, as well as the structure and DNA binding of the C- terminal GRF zinc finger domain. We are working to understand how the unique architecture of NEIL3 enables its recruitment and positioning at convergent forks, and how the catalytic domain is capable of accessing a conformationally constrained crosslinked nucleotide. Our work addresses critical gaps in knowledge related to how the intrinsic activities of these enzymes are regulated by interactions within the replisome.

NIH Research Projects · FY 2025 · 2020-04

PROJECT SUMMARY Rates of anxiety and depression in youth are substantial, causing a major unmet need for effective interventions. Although some progress has been made in preventing these internalizing problems in adolescents, further research is needed that specifically targets theoretically and empirically supported risk processes. An important and salient risk factor found to increase the likelihood of anxiety and depression is negative affectivity – a partially heritable trait propensity to experience and express more frequent, intense, and enduring aversive emotional states. The proposed randomized controlled prevention trial builds on our finding from our longitudinal study that elevated levels of negative affectivity during adolescence prospectively predicted internalizing disorders in early adulthood (Zinbarg et al., 2016); moreover, this relation was mediated by changes in momentary negative affect (mNA) measured with ecological momentary assessment (EMA) (Adam et al., 2018). The first phase (R61) of the proposed selective prevention trial will test whether an app-based, coach-supported mindfulness intervention as compared to an assessment-only control reduces momentary negative affect, measured with ecological momentary assessment (EMA), in 120 adolescents (age 12-16) at- risk based on their having high levels of trait negative affectivity. EMA will be used to measure average daily mood, (the “Target”) collected six times a day across three days at pre-, mid-, and post- intervention. “Target” engagement will be defined as a medium effect size (>.40) in the comparison of youth randomized to MBI versus control on the target – momentary negative affect – at post-test, adjusting for pre-test levels. We also will assess the dose-response relation by testing the association between number of sessions and exercises completed with changes in momentary negative affect and weekly mood ratings. In the second phase (R33), we will conduct a replication trial with a new sample of 360 at-risk (i.e., high trait negative affectivity) youths (ages 12-16) randomized to one of three conditions – MBI, a nonspecific control, or an assessment-only control. Youth will be evaluated with regard to the target (i.e., mNA), internalizing symptoms and disorders, and functioning (e.g., social, academic) at baseline and post-intervention (R61 and R33), and at a 6-month follow-up (R33). Finally, in the R33 we will test if significant reductions in momentary negative affect are associated with improvements (or less worsening) in internalizing symptoms and fewer onsets of internalizing disorders.

NIH Research Projects · FY 2025 · 2019-09

Most health research on sexual and gender minority (SGM) populations focuses on younger people. Only 7.6% of NIH-funded SGM-related projects examine aging, despite 27% of SGM individuals being over age 50. The limited data we do have reveal significant disparities in health and aging outcomes among midlife and older SGM adults, including earlier and more severe cognitive decline, increased risk of Alzheimer’s disease and related dementias (ADRD), and earlier mortality, likely driven by chronic exposures to minority stressors over the life course. There is an urgent need for high-quality data on midlife and older SGM adults to identify intervention opportunities and address upstream determinants of health. Our current project, the LGBTQ+ Social Networks, Aging, and Policy Study (QSNAPS; R01AG063771), successfully recruited and retained the largest cohort of older SGM adults in the US South (N=1256; Age 50-76). QSNAPS data have provided valuable insights into health and aging outcomes among midlife and older SGM adults, emphasizing the importance of SGM-affirming healthcare. With NIH pilot and supplement funds, we collected for the first time biomarkers of aging and objective measures of cognitive function among self-identified SGM adults. Public release of all data is in process. This renewal project strategically expands research on midlife and older SGM adults by continuing to provide high-quality, longitudinal data and expanding biomarker and cognitive testing data in this NIH health disparity priority population. We will reinterview QSNAPS participants (N=1256) and recruit a 30% refresher sample (N=375), oversampling underrepresented groups to improve representation (Aim 1). Waves 4 and 5 will be publicly available and linkable to prior waves, capturing up to 8 participant life-years. All Wave 4 participants will be invited to participate in cognitive testing and biomarker collection, both of which we have piloted in this sample to ensure feasibility, acceptability, and the presence of preliminary associations consistent with hypotheses. Using cognitive testing data at Waves 3, 4, and 5, we will examine differences in cognitive function and change by sexual orientation and gender identity and test the effects of SGM-specific stressors on cognitive functioning and change over a 4-year period (Aim 2). We will also collect biomarkers linked to chronic stress and inflammation (CRP, IL-6, TNF), and construct surrogate markers using DNA methylation data that reflect global measures of biological aging, inflammation, and neurodegeneration using the Tasso+ self-collection device. We will use biomarker data to test the effects of SGM-specific stressors on chronic stress and inflammation, biological aging, and neurodegeneration, thereby advancing minority stress research by directly testing the proposed physiological mechanisms (Aim 3). Overall, this study addresses the critical need for high-quality SGM-inclusive aging studies and uses innovative methods to collect--for the first time--objective measures of cognitive function and biomarker data among a large panel of self-identified SGM adults.

NIH Research Projects · FY 2025 · 2019-09

Project Summary / Abstract The goal of our laboratory is to interface challenging and thought-provoking organic chemistry with issues of plausible biological application. A primary goal for the next five years is to develop new molecular probes and leverage these molecules to advance our understanding of human disease. Critical to the role which the laboratory plays at Vanderbilt University, and within larger concerns of glycobiology, is problem selection. On the chemistry side, we identify problems of a complexity level conducive to new and significant learning experiences. At the level of biology, we select difficult problems that may be subject to accelerated advance if the necessary chemistry can be developed. Taken together, the overall vision for our program is to apply a human health driven approach to developing robust and reproducible reactions and synthesis strategies to interrogate clinically relevant diseases.

NIH Research Projects · FY 2026 · 2019-09

PROJECT SUMMARY Proteins fold into their 3-dimensional shape with the help of chaperones and other protein folding factors, which together comprise the proteostasis network (PN). During the protein quality control process, transient binding interactions between individual client proteins and proteostasis factors mediate folding into native functional structures, thereby ensuring trafficking to the correct cellular destination or facilitating degradation of detrimental misfolded states. Consequently, imbalances in interactions between proteostasis factors and client proteins result in quality control defects that lead to diverse protein misfolding diseases, including highly prevalent neurodegenerative diseases such as Alzheimer’s and Parkinson’s Disease, and loss-of-function diseases such as Cystic Fibrosis. The folding, maturation, and trafficking of large multi-domain and multi-subunit proteins is a complex and highly client-specific process that depends on engaging the appropriate component of the PN at the correct time. Many proteostasis dependencies for individual client proteins are known, but little is understood about the engagement order and whether correct sequential interactions are required for proper folding and trafficking. We have utilized the power of chemical biology and quantitative proteomics to develop a new tool, time-resolved interactome profiling, to interrogate the dynamics of the interaction between disease-associated protein variants and the PN. We examined the coordination requirement of different proteostasis pathways as they affect protein secretion (thyroglobulin) and loss-of-function protein misfolding (cystic fibrosis transmembrance conductance regulator – CFTR). Loss of secretion of destabilized thyroglobulin variants, a thyroid prohormone, is a leading cause of congenital hypothyroidism while misfolding and pre-mature degradation of CFTR variants is the primary cause of Cystic Fibrosis. We have identified three projects that leverage the utility of our approach and build upon the groundwork laid by our previous findings. These include: 1) Determining the contribution of degradation factors to secretion loss of misfolded thyroglobulin variants. 2) Elucidating the proteostasis dynamics of divergent multipass membrane proteins, and 3) Enhancing the time-resolution of our time-resolved interactomics approach to capture early ER translocation and folding dynamics. Results from our studies will provide significant new insights into how the dynamics of protein folding and trafficking pathways can be manipulated therapeutically for disease intervention.

NIH Research Projects · FY 2026 · 2019-08

Project Summary In all eukaryotic cells, export of mRNA from the nucleus to the cytoplasm is an essential step in gene expression. As such, mRNA nuclear export is fundamental for all cellular activities and has been linked to various diseases when it is dysregulated, such as in cancer, neurological disorders, and viral infections. Our research focuses on elucidating the mechanisms and regulation of mRNA export under physiological and pathological conditions. Within the nucleus, newly synthesized mRNAs are processed and packaged with proteins to form ribonulceoprotein particles (mRNPs). The core machinery that facilitates the assembly of export-competent mRNPs is the TREX (TRanscription/EXport) complex. Through the enzymatic activity of its ATPase subunit Sub2/DDX39B, TREX facilitates loading of export-specific factors including the export receptor NXF1•NXT1 to form export-competent mRNPs. We have recently elucidated the architecture of the TREX complex and defined key steps of Sub2/DDX39B’s activity. These findings provide a solid foundation for us moving forward to investigate fundamental aspects of mRNA export that remain poorly understood. In this grant period, we aim to identify and structurally characterize the physical and functional interaction network that connects different steps of mRNA export and coordinates the export process with other nuclear events. We envision that these molecular connections spatiotemporally regulate the ATPase- driven mRNP assembly and establish selective export of mature transcripts. The resulting biochemical and structural knowledge will generate hypotheses that we will test at the cellular level. In addition, we aim to investigate how viruses exploit host mRNA export to facilitate their replication. We recently have elucidated the molecular basis for how influenza A virus and SARS-CoV-2 target NXF1•NXT1 to block host gene expression. We will continue to study the various mechanisms that influenza A virus uses to exploit host mRNA export factors. Together, our work will provide mechanistic insights into the mRNA export pathway and may offer novel therapeutic opportunities for viral infections.

NIH Research Projects · FY 2026 · 2019-05

Summary Cytokinesis, the physical separation of one cell into two daughter cells, is the final stage of cell division, and although it is the least well understood, it is central to development and tissue homeostasis. Correctly timing cytokinesis so that it occurs only after chromosome replication and segregation is necessary to prevent catastrophic genomic instability, and accordingly, cytokinesis is strictly regulated in concert with other cell cycle events. Using a powerful model organism, the fission yeast Schizosaccharomyces pombe, my lab has conducted pioneering research to identify proteins essential for cytokinesis and to learn how the myriad proteins that comprise the cell division machinery are coordinated to ensure the exquisite spatial and temporal control of cell division. We propose to continue our work pursuing fundamental questions in this field using a multi-disciplinary approach in two directions. In one direction, we will tackle how CK1 protein kinases are able to inhibit cytokinesis during a delay in spindle assembly by pursuing their regulation. We will determine whether CK1 activity is regulated by cell cycle cues. Understanding CK1 regulation in the context of the mitotic checkpoint will establish general mechanisms of regulation for this enzyme family, which are conserved, multifunctional kinases with roles in numerous human diseases. In a second direction, we will advance our understanding of the assembly and architecture of the contractile ring using a combination of microscopy, biochemistry, and genetics. We will continue to build our knowledge of the major scaffold of the contractile ring, the F-BAR protein Cdc15, by defining how it oligomerizes on the plasma membrane, and how other contractile ring components including the phosphatase calcineurin are organized on the Cdc15 scaffold. These focused mechanistic studies will be complemented with proteomic and genetic screens designed to establish a functional interaction network of contractile ring components. Together, these studies will have a major impact for understanding how cytokinesis is orchestrated in eukaryotic species from yeast to humans.

NIH Research Projects · FY 2026 · 2019-04

PROJECT SUMMARY Thousands of abasic sites form daily in each of our cells. Many types of environmental toxins that cause alkylation or oxidation of DNA bases to form N7-guanine adducts and 8-oxoguanine induce abasic sites. For example, N-nitrosamines that are found in foods, detergents, solvents, plastics, and agricultural chemicals as well as chemicals like carbon tetracholoride, potassium bromate, and chloroform that induce oxidative stress all increase the frequency of abasic sites in DNA. Failures in managing this ubiquitous form of DNA damage can cause a variety of diseases including cancer. The known mechanisms of repair require an intact DNA duplex; however, abasic sites form more readily in single-stranded DNA where they are impediments to replicative polymerases. We recently discovered a new pathway that detects and process abasic sites in single-stranded DNA. This pathway utilizes HMCES (hydroxyl-methyl cytosine embryonic cell specific) to detect and shield these abasic sites from deleterious processing. HMCES contains an evolutionarily conserved domain (SRAP) that binds DNA and forms a covalent crosslink to abasic sites. This DNA-protein crosslink prevents endonucleases from cleaving the single-stranded DNA, thereby preventing double-strand breaks. In this proposal we will further characterize how this pathway acts to maintain genome stability, and more broadly define how abasic sites are tolerated and repaired in the context of DNA replication. We will utilize state of the art biochemical, genetic, and structural approaches in human cells and cell extracts. Completing these studies will provide a mechanistic understanding of how cells cope with a ubiquitous form of DNA damage generated by important environmental genotoxins.

NIH Research Projects · FY 2026 · 2019-04

Project Summary The premise of this application is that discovering the basic mechanisms and functions of replication stress signaling pathways will contribute to the development of effective cancer therapies. Specifically, our goal is to understand how the ATR (ATM and rad3-related) kinase promotes genome stability and how its inhibition could be useful as a cancer therapy. ATR functions at the apex of DNA damage and replication stress response pathways that are needed every cell division cycle to promote the complete and accurate duplication of the genome. Many cancer cells are highly dependent on ATR function for proliferation and viability because of elevated levels of oncogene-induced replication stress and mutations in other genome maintenance pathways. Inhibiting ATR hypersensitizes cells to DNA damaging chemotherapies and targeted agents like PARP inhibitors. Thus, ATR may be a useful drug target and ATR inhibitors are currently in clinical trials in a variety of cancer settings. We previously defined how ATR is activated, identified critical targets to control DNA replication and cell division, and defined synthetic lethal opportunities where ATR inhibitors could be useful. In this proposal we will extend these studies to examine specific mechanisms of ATR action that promote the survival of cancer cells. We will define mechanisms for how ATR regulates DNA damage tolerance pathways, use unbiased approaches to expand our understanding of ATR-dependent regulation of replication stress responses, and study the unique functions of individual ATR activating proteins including ETAA1. This is a focused proposal aimed at understanding the most important and least understood aspects of ATR function. Specific hypotheses and innovative concepts based on preliminary data are tested using advanced biochemical and genetic approaches. In addition, the aims provide opportunities for unexpected discoveries about the mechanisms that maintain genome integrity and the mechanisms of action of ATR pathway inhibitors.

NIH Research Projects · FY 2025 · 2018-12

PROJECT SUMMARY Natural product therapeutics remain critically important in the treatment of cancer, with most patients diagnosed with cancer receiving natural product-based chemotherapy, such as microtubule disrupting agents and DNA intercalators. Select chemotherapeutic small molecules, the majority of which are natural products, have an additional therapeutic benefit in their ability to stimulate a productive form of natural immunity against the cancer cells that they injure or kill. For example, chemically induced damage associated molecular pattern expression can recruit antigen presenting cells to phagocytose damaged cancer cells and display cancer cell antigens to prime and activate T cells for adaptive immunity. Notably however, natural product induced anti-tumor immunity properties of these select compounds were only discovered subsequent to their clinical application. We hypothesize that the pathways by which such immunogenic natural products injure and kill cancer cells determines the production of damage associated molecular patterns and ensuing innate and adaptive immune responses against treated cells. We propose to develop and apply a high throughput, multiplexed, single cell chemical biology assay platform for the discovery of chemically induced antitumor immunity. Using this system, we will map the associations between regulated cell death and injury signaling in treated cells to functional cellular markers of immunogenicity for several classes of known and new natural products. This goal will be accomplished through three aims: (1) Discover and characterize secondary metabolites via regulated cell death and injury cytometric phenotypes; (2) Define the relationships between cell injury and death signaling phenotypes and damage associated molecular patterns, and immunogenic cell signaling in cancer and immune cells; (3) Validate chemical agents inducing immunogenic cell injury and death via antigen cross priming assays and deep cellular profiling using flow cytometry. Relevance: The successful completion of the proposed research is relevant to human health because it will provide methods to accelerate the identification of potential anticancer natural products, which have had and continue to have a large positive impact on human health. Furthermore, the discovery of the multiplexed cell-targeting immuno-oncological structure-activity relationships within newly discovered compounds and known but not commercially available compound families, may provide new targeted therapeutics, with greater efficiency and reduced clinical toxicity.

NIH Research Projects · FY 2026 · 2018-09

Project Summary Every cell division requires the faithful duplication of genetic material from mother cell to daughter cell. Defects in the proper execution of the DNA replication program can directly result in the genome instability that is a hallmark of nearly all cancer cells. To ensure genome stability, the machinery responsible for DNA replication must be highly regulated. There must be enough flexibility built into the DNA replication program, however, to accommodate the dramatic changes in cell cycle and S phase progression that occur during the development of an organism. Understanding this regulation is imperative to determine how genome stability is maintained during development. We utilize the powerful developmental systems available in Drosophila to identify key regulators of the DNA replication program, with the ultimate goal of delineating the molecular underpinnings responsible for regulating genome duplication and stability during development. Our main focus is understanding how replication initiation is regulated to ensure genome stability. We have defined three key projects where the strength of our system positions us to make key insights. These include: 1) understanding the functional relationship between nuclear pore proteins and the Origin Recognition Complex (ORC). 2) Defining the key targets and molecular mechanisms Rif1 uses to influence replication initiation. 3) Identifying and characterizing posttranslational modifications of ORC that affect replication initiation.

NIH Research Projects · FY 2025 · 2018-08

Project Summary Led by a multi-PI team of a cell biologist and a biophysicist, this project is a renewal of our investigation into the cellular detection of and responses to wounds. For our model system, we use the Drosophila pupal notum, a diploid epithelial monolayer, and we wound it by laser ablation. Although the tissue is diploid, the region at and near the wound margin is dominated by giant syncytial cells. The origin, function, and fate of these syncytial cells are all unknown. Using live imaging, we have found that the giant syncytia are formed via cell-cell fusion of multiple diploid cells. These fusions occur within ~20 minutes of wounding and the resulting syncytial cells migrate more quickly and close wounds faster than diploid cells. By the end of tissue repair, most of these giant syncytia are eliminated from the epithelium. Interestingly, we have found that the amount of cell fusion and syncytia formation depends on the mode of wounding. We will compare wound healing behaviors in wounds that lack syncytia and those that have syncytia to investigate how these giant cells increase the rate of wound closure (Aim 1). In Aim 2, we will investigate how wounds induce mononuclear diploid cells to fuse into syncytia. In Aim 3, we will analyze the long-term fate of these syncytia, which appear to die by apoptosis and extrusion as wound closure is ending. Syncytial and polyploid cells have been observed in other organisms and tissues in response to wounds, but our system is the first to make a detailed analysis of their formation, contribution, and elimination possible using live imaging. Cells involved in wound-healing generally share behaviors with tumor cells, and the wound- induced giant syncytial cells may represent the wound equivalent of Giant Polyploid Cancer Cells, a syncytial cell type found in many cancers. Giant Polyploid Cancer Cells are malignant, resistant to all therapies, and appear to be a major source of tumor cells fueling metastasis and relapse. We expect that our studies into the adaptive functions of wound-induced syncytia will be important for understanding the biology, origin, and potential therapies for maladaptive Giant Polyploid Cancer Cells.

NIH Research Projects · FY 2025 · 2018-08

PROJECT SUMMARY Failure to replicate even a short stretch of DNA leads to chromosome missegregation and activation of error prone DNA repair pathways. It is therefore crucial to ensure completion of DNA synthesis. However, the completion of DNA synthesis is not under surveillance by cell cycle checkpoints. Additionally, while defects in earlier stages of replication can be overcome by new initiation events, this is essentially impossible when short stretches of unreplicated DNA remain. Thus, exquisitely effective DNA synthesis mechanisms are needed to ensure the completion of DNA synthesis. The long-term goal of my lab is to elucidate and understand these mechanisms. In the current proposal, we focus on the following mechanisms that normally support completion of DNA synthesis, as well as mechanisms that restart DNA replication in response to DNA damage: (1) Replication termination occurs when two converging replication forks meet on the same stretch of DNA and is how DNA synthesis is normally completed. We recently found that topological stress can cause converging replication forks to stall and that this is overcome by both topoisomerase II-dependent and - independent mechanisms. However, it is unclear why other topoisomerases cannot compensate for loss of topoisomerase II and how these different mechanisms are efficiently engaged to ensure rapid fork convergence. We will determine the roles that different topoisomerases play during DNA replication and how their respective roles contribute to replication termination. We will also investigate how the different proteins that promote fork convergence recognize their targets to ensure that obstacles to termination are efficiently overcome. (2) Replication fork reversal and Nascent Strand Degradation (NSD) are thought to allow replication forks to bypass DNA lesions in an essentially error-free manner. However, it is unclear how exactly NSD contributes to restart of reversed forks. We recently developed a new approach to induce efficient fork reversal and NSD and identified new steps involved in this process. We will leverage our approach and insights to determine the role that NSD plays in restart of reversed forks. (3) Break-Induced Replication (BIR) restarts DNA synthesis from a double-stranded DNA end. However, it is unclear how BIR ultimately leads to completion of DNA synthesis and what the full set of required proteins is. Additionally, Mitotic DNA Synthesis (MiDAS) is a BIR-like process that operates during mitosis but it is unclear how the choice between MiDAS and BIR is determined. We have developed an approach to induce and monitor BIR, which makes us uniquely equipped to address these questions. We will determine how BIR completes synthesis and the proteins involved. We will also determine how the choice between BIR and MiDAS is determined.

NIH Research Projects · FY 2025 · 2018-08

SUMMARY Background and key gaps in knowledge: Mitochondria serve as hubs coordinating diverse aspects of cellular signaling, metabolism and inter-organelle dynamics. Our work during the ESI MIRA funding period has revealed multiple roles for MCL-1, an anti-apoptotic BCL-2 protein, in the regulation of mitochondrial morphology and cell state. Detailed mechanistic studies are still needed to understand this most basic aspect of MCL-1 function. The non-apoptotic function of MCL-1 converges and relies on the mitochondrial dynamics machinery. The dynamic nature of the mitochondrial network determines the ability of this organelle to regulate a variety of cellular processes that are tightly linked to cell fate and identity. Mitochondria coordinate these functions by coordinated remodeling, as well as by the physical and functional interaction with other organelles, such as peroxisomes, which also undergo remodeling to coordinate their function. The critical need for controlled mitochondrial and peroxisomal morphology in the maintenance of cellular homeostasis is highlighted by the wide spectrum of neurodevelopmental diseases associated with mutations in the regulators of organelle dynamics. We will combine our expertise in organelle and stem cell biology with state-of-the-art approaches to uncover the mechanisms by which mitochondrial and peroxisomal dynamics maintain neurogenesis. Description of recent progress by PI: In pluripotent stem cells, we reported that MCL-1 is required for maintaining an apoptotic priming state and pluripotency through its regulation of mitochondrial morphology (Rasmussen et al., 2018). In differentiated cells, we found that MCL-1 is also required to maintain cell identity (Rasmussen et al., 2020; Cleveland et al., 2021). This function which is shared with other members of the BCL- 2 family (Joshi et al., 2020) requires the fine-tuned coordination with the mitochondrial dynamics machinery (Rasmussen et al., 2018 and 2020; Joshi et al., 2020). The fundamental role of organelle remodeling in driving cell fate determination, led us to examine the impact of abnormal mitochondrial and peroxisomal fission on neurogenesis (Baum et al., 2020; Robertson et al., 2022, Robertson et al., 2023 in press), using an iPSC-based discovery platform optimized to study organelle biology (Romero-Morales et al., 2019, 2022a, 2022b). Overview of future research program: We propose to build upon our findings from the ESI MIRA by taking a multi-faceted approach aimed at three areas: 1) Continue elucidating how MCL1 controls stem cell identity and differentiation by regulating mitochondrial dynamics and metabolism. 2) Modeling the contribution of mitochondrial and/or peroxisomal fission to cellular transitions during neuronal differentiation. 3) Finally, we will identify the metabolic switches that are engaged by mitochondrial and peroxisomal remodeling during neuronal differentiation. Detailed elucidation of these mechanisms will reveal connections between organelle dynamics and cell fate as well as the relationship of these processes to the neuronal dysfunction associated with rare mitochondrial and peroxisomal diseases.

NIH Research Projects · FY 2025 · 2018-05

This proposal is a renewal of a successful T32 training program entitled “Vanderbilt Interdisciplinary Training Program in Alzheimer’s Disease.” This training program provides robust interdisciplinary, translational research training for biomedical PhD students and post-doctoral fellows in Alzheimer’s disease and related dementias. The program capitalizes on the rich institutional resources of Vanderbilt University, Vanderbilt University Medical Center, and Meharry Medical College and the interdisciplinary research strengths of our training faculty. Established in 2018, this T32 program aims to position the next generation of basic, translational, and clinical scientists to advance towards independence while making major contributions to the understanding of Alzheimer’s disease and its intersection with neurodegeneration and concomitant pathologies that contribute to clinical manifestation and decline (e.g., cerebral small vessel disease). During the initial award cycle, we established our training curriculum, built awareness of our program across campus (resulting in a steady growth of competitive nominations), and appointed 20 total trainees (9 PhD students, 11 post-doctoral fellows). Trainee outcomes are strong with all program graduates retained in aging or neuroscience research-related careers and 7 trainees receiving independent funding. Concurrent with the launch of this T32 training program, we also launched our new National Institute on Aging-funded Vanderbilt Alzheimer’s Disease Research Center. This new Center grant has resulted in extensive expansion of our Alzheimer’s disease and related dementias research activities and increased the richness of our institutional infrastructure, data resources, intellectual neighborhood, and training opportunities. Through individualized mentorship from faculty and peer networks, an integrated curriculum, and involvement in cutting-edge research, our program will continue to emphasize basic science and clinical fundamentals of Alzheimer’s disease and related dementias. Trainees will continue to develop essential research and professional skills for building a successful career along with a deep appreciation for rigor, reproducibility, and responsible conduct of research. Participation in a high-quality interdisciplinary research experience will synthesize the trainees’ knowledge base, research and professional skill set, and appreciation of research ethics. Each trainee’s individualized training plan will be augmented by additional institutional and programmatic resources to foster academic excellence and scientific innovation during these early, formative periods of professional development.

NIH Research Projects · FY 2026 · 2018-04

Summary Microtubules (MTs) are dynamic biopolymers, which serve as major highways for intracellular transport. MT networks are critical for cell physiology, and their disturbance underlies many human diseases. My laboratory studies global mechanisms allowing MTs to perfectly attribute to specific cellular functions. MT-dependent transport arranges multiple cell components at the same time. To address these complex requirements, MT geometry, molecular motor affinity, and/or MT association with to other cell components are tightly regulated. MT network geometry changes depending on the sites of new MT outgrowth (the MT-organizing centers, or MTOCs), local stabilization/disassembly of MTs, and MT anchoring to other structures. Furthermore, the affinity of individual MTs to molecular motors can be modulated to affect intracellular transport. Finally, MTs can scaffold proteins or be cross-linked with other cytoskeletal components. All those mechanisms that tailor MT organization to distinct cell functions can respond dynamically to cell-signaling inputs and the physiological context. Together, molecular regulation and functional specialization of MT networks comprise a global field in basic cell biology with numerous unanswered questions. My research program’s long-term goals include defining: how interphase MT networks are built and regulated; specific mechanisms tailoring MT biochemistry and geometry to specific cellular needs; the methods whereby MTs collaborate with other cellular systems to build intracellular space; and, how MTs switch their functional loads between distinct tasks to arrange integral cell architecture under changing signaling conditions. Since April 2018, the NIGMS MIRA funding mechanism has been an invaluable resource allowing us to explore these basic, fundamental biological problems. We have published several central advances toward our global and interactive goals. Among other findings, we brought a new mechanistic understanding of Golgi-derived MT networks (GDMTs, which were identified in our prior studies); described novel, surprising functions for MT- associated proteins (MAPs) tau, CLASP2, and CAMSAP2; utilized collaborations with experts in computational modeling for deep understanding of MT functions in secretory trafficking and actin cytoskeleton dynamics; and, discovered a previously overlooked, physiologically important Golgi complex behavior in the cell cycle. In the next five years, I will extend mechanistic and functional insights in two broad directions of my program’s extant NIGMS-funded research. (I) We will determine how the versatility of MT functions is tuned by multifunctional MAPs, focusing on (a) secretory trafficking through the Golgi axis and (b) the organization of the actin cytoskeleton. Initial studies will evaluate the roles of MT regulators CLASPs and a MT-stabilizing tumor suppressor RASSF1A. (II) We will expand our studies of MT-dependent Golgi positioning to dissect (a) molecular mechanisms driving this process and (b) the significance of Golgi relocation in distinct cell cycle stages.

NIH Research Projects · FY 2025 · 2018-02

SUMMARY Nucleotide excision repair (NER) protects human cells by removing harmful DNA damage, but repair of damaged DNA by NER can reduce the efficacy of some antitumor drugs such as cisplatin. NER genes are frequently missense mutated in cancers and decreased expression or loss of function mutation of NER genes ERCC1 and ERCC2, respectively, has been shown to correlate with improved patient outcomes after cisplatin treatment. This proposal investigates the hypothesis that reduced NER capacity arising from tumor mutations correlates with greater sensitivity to platinum (Pt) agents. It focuses on the NER scaffolding protein XPA, which is required for proper assembly and organization of the NER machinery. XPA is an “Achilles Heel” of NER because it interacts with the DNA substrate and nearly all core NER proteins. Our recent publications (i) show NER is suppressed by XPA mutations that inhibit interaction with its partner scaffold RPA, and (ii) identify XPA mutations from tumor genomes that disrupt NER, including some that our current work suggests are highly likely to disrupt the interaction of XPA with RPA. Thus, XPA represents the ideal factor to investigate the hypothesis that reduced NER capacity correlates with sensitivity to DNA damaging agents. Aim 1 will test the hypothesis that missense mutations in XPA can lead to NER defects that reduce repair capacity and sensitize tumor cells to Pt agents. XPA mutations will be screened for reduced NER capacity using a high throughput reporter assay to select those for which NER deficiency will be further characterized in cells expressing XPA mutants. We will then determine the mechanism of their dysfunction and test their sensitivity to Pt agents. Aim 2 will use a fragment-based discovery approach to develop small molecule inhibitors that disrupt the XPA-RPA interaction to enable further tests of the correlation between NER capacity and sensitivity to Pt agents. A highly curated library of small molecular fragments will be screened by NMR and the binding location and orientation of ‘hits’ will be defined by X-ray crystallography. After cycles of optimization involving structural analysis, design, and evaluation, linked fragment compounds will be validated for physically inhibiting XPA-RPA interaction, suppressing NER, and eliciting sensitivity to Pt agents in cancer cell lines. Together, these aims will not only test the correlation between NER deficiency and sensitivity to Pt agents, but also generate tool compounds that lay the foundation for testing the therapeutic value of inhibiting NER. They will also provide valuable insights to move closer to the use of Pt sensitivity predictors in the clinic and explore how NER inhibition affects sensitivity to other DNA damaging agents. Ultimately, we seek to understand how the tumor genomic landscape predisposes cancer cells to drug sensitivity to enable identification of patient tumors that will be sensitive to DNA damaging agents alone or require combinatorial treatment with NER inhibitors.

NIH Research Projects · FY 2025 · 2018-02

Type II topoisomerases are ubiquitous enzymes that are required for proper chromosome structure and segregation and play important roles in DNA replication, transcription, and recombination. These enzymes relax DNA and remove knots and tangles from the genetic material by passing an intact double helix (transport segment) through a transient double-stranded break that they generate in a separate DNA segment (gate segment). Humans encode two closely related isoforms of the type II enzyme, topoisomerase IIα and topoisomerase IIβ. Topoisomerase IIα is essential for the survival of proliferating cells and topoisomerase IIβ plays critical roles during development. However, because these enzymes generate requisite double-stranded DNA breaks during their crucial catalytic functions, they assume a dual persona. Although essential to cell survival, they also pose an intrinsic threat to genomic integrity every time they act. Beyond their critical physiological functions, topoisomerase IIα and IIβ are the primary targets for some of the most active and widely prescribed drugs currently used for the treatment of human cancers. These agents kill cells by stabilizing covalent topoisomerase II-cleaved DNA complexes (cleavage complexes) that are normal, but fleeting, intermediates in the catalytic DNA strand passage reaction. When the resulting enzyme- associated DNA breaks are present in sufficient concentrations, they can trigger cell death pathways. Anticancer drugs that target type II enzymes are referred to as topoisomerase II poisons because they convert these indispensable enzymes to potent physiological toxins that generate DNA damage in treated cells. Although topoisomerase IIα and IIβ are important targets for cancer chemotherapy, they also have the potential to trigger specific leukemias. For example, a small percentage of patients with cancer or multiple sclerosis who are treated with the topoisomerase II-targeted drug mitoxantrone go on to develop acute promyelocytic leukemias (APLs) with 15:17 translocations. Despite the importance of type II topoisomerases in cell growth and cancer, we still have much to learn about how the human enzymes function and interact with DNA and anticancer drugs. Thus, this proposal will further define the catalytic mechanism of type II topoisomerases and examine how enzyme activity is regulated in the cell. It also will define mechanisms by which established and novel topoisomerase II-targeted agents, environ- mental chemicals, and natural products increase levels of enzyme-mediated DNA breaks or inhibit enzyme activity and determine the cellular consequences of topoisomerase II poisons. The primary research models for this study will be human topoisomerase IIα and IIβ, cultured human cells, and Xenopus laevis egg extracts. Gyrase and topoisomerase IV from Escherichia coli and Bacillus anthracis will be used as counterpoints to mechanistic experiments and Mycobacterium tuberculosis gyrase will be used to assess relationships between the mechanisms of action of drugs targeted to prokaryotic and eukaryotic type II enzymes.

NIH Research Projects · FY 2026 · 2017-08

1) Background and key gaps in our understanding. Cellular force generation drives processes vital for eukaryotic life, including cell division, cell migration, and muscle contraction. Thus, the basic principles underlying cellular force generation are central to both developmental biology and the progression of force-dependent diseases like cancer. Cells generate force by assembling cellular contractile systems. Contractile systems emerge from the collective action of individual components within a larger molecular assembly. Our first investigation into cellular contractile systems focused on the class of molecular motors responsible for generating contractile forces, myosin II. The force-generating form of myosin II is a filament. A given myosin II filament can contain multiple, distinct myosin II paralogs (i.e., hetero-filaments), wherein each individual myosin II paralog has unique biophysical properties. Previously, the specific function(s) of myosin II-containing hetero-filaments within contractile systems was unknown. Our work during the ESI MIRA funding period has revealed multiple roles for hetero-filaments in the context of cell division and muscle contraction. Future work should address how hetero- filaments are regulated, influence mechanical output, and cooperate with other contractile system components. 2) Description of recent progress by the PI. In non-muscle cells, we reported that hetero-filaments serve multiple functions. Myosin IIA filaments seed the formation of myosin IIB filaments in both interphase and mitosis/cytokinesis (Fenix et al. 2016, Taneja et al., 2020). The presence of myosin IIA in hetero-filaments drives cortex tension (Taneja et al. 2020) and is regulated by both myosin IIA turnover and by phosphorylation of the myosin IIA regulatory light chain (Taneja and Burnette 2019, Taneja et al. 2021). Meanwhile, the presence of myosin IIB in hetero-filaments stabilizes the cell cortex during mitosis/cytokinesis and regulates cytokinetic fidelity through multiple mechanisms (Taneja et al. 2020). Within cardiac muscle cells, we reported that NMIIA/NMIIB hetero-filaments seed filaments of the muscle-specific myosin paralog, β myosin II, and are found exclusively in the sarcomere precursors known as muscle stress fibers. We also experimentally demonstrated that muscle stress fibers directly give rise to sarcomeres (Fenix et al. 2018, Taneja, Neininger et al. 2020). 3) Overview of future research program. Here, we propose to build upon our findings from the ESI MIRA by taking a multi-faceted approach aimed at three areas: A) We will continue elucidating how specific molecular components of myosin II filaments, as well as those of other contractile system proteins like α-actinin and formins, regulate cellular force production. B) We will determine the roles, and specific differences in function, of the non- muscle and muscle paralogs of myosin II, α-actinin, and potentially other sarcomeric proteins during both sarcomere formation and mitosis/cytokinesis (we have found that some muscle paralogs re-localize from the sarcomere to the cortex during cell division). C) Finally, we will use zebrafish embryos to test the hypotheses that come out of our in vitro experiments in vivo.

NIH Research Projects · FY 2026 · 2017-04

PROJECT SUMMARY: The evolutionarily conserved Wnt pathway plays a critical role during metazoan development and stem cell maintenance in the adult. Mutations in the Wnt pathway leading to its misregulation in humans have been shown to contributes to both developmental defects and cancers. In the latter case, over 90% of all non-hereditary forms of colorectal cancers are initiated by mutations in the Wnt pathway leading to its inappropriate activation. There are no FDA-approved drugs that inhibit the Wnt pathway, which is likely due to our incomplete understanding of the mechanism of Wnt signaling and the lack of suitable drug targets. The primary focus of my lab is to understand the biological function of the Wnt signaling pathway by deciphering its molecular mechanism and identifying new Wnt components. Our ultimate goal is to use this information to understand how deregulation of the Wnt pathway can lead to Wnt- driven diseases in humans. Over more than a decade, my lab has made significant contributions to our fundamental understanding of Wnt signaling. These breakthroughs were accomplished via the development of the first biochemical system (using Xenopus egg extract), the reconstitution of active purified proteins, the development of the first mathematical model (Lee-Heinrich model), and the identification of small molecule inhibitors (CK1 agonists), one of which has been designated by the FDA as an orphan drug for a familial precancerous disease (familial adenomatous polyposis). Our current work focused on determining the role of the USP46/UAF1/WDR20 deubiquitinase complex in regulating LRP6 receptor turnover, the mechanism by which APC regulates Wnt receptor activity, the role of the -catenin degradation complex in mediating bistable pathway behavior, and the roles of USP47 and STK38, in regulating Wnt signaling. In our renewal application, we now propose to 1) reconstitute LRP6 ubiquitylation and identify its evolutionarily conserved E3 ligase, 2) understand the basis for signal compensation upon Wnt receptor loss in APC mutant cells, 3) provide evidence for the role of bistability in generating morphogenetic signaling, and 4) uncover the roles of the STK38 and TRIP12 in nuclear Wnt signaling.

NIH Research Projects · FY 2026 · 2017-02

SUMMARY During differentiation, enterocytes build an extensive apical array of microvilli known as the brush border, which serves to amplify the plasma membrane surface area available for nutrient absorption. An individual microvillus is simple in structure, consisting of a supporting core bundle of ~25 actin filaments that protrudes from the apical surface wrapped in membrane. In addition to serving as the sole site of nutrient uptake, brush border microvilli also provide an anchoring point for the glycocalyx and regulate interactions with luminal microbes. Although the brush border serves as the primary functional interface of the intestinal tract, mechanisms that drive the timely formation of microvilli during enterocyte differentiation remained unclear until recently. During our first funding period, we discovered several factors that control actin filament polymerization during microvilli formation, including the IRTKS/EPS8 complex. However, building stable microvilli also requires that actin filaments are organized into core bundles, which exhibit flexural rigidities high enough to deform the apical surface. How nascent enterocytes coordinate the fundamental activities of actin filament polymerization and bundling in space and time to build stable microvilli remains unknown. In recent preliminary studies, we used a proximity labeling approach to identify proteins within ~20 nm of IRTKS/EPS8 puncta during microvillus assembly; this screen led to our exciting discovery of Mitotic Spindle Positioning (MISP) as a new actin filament bundling protein in the brush border. MISP is expressed along the full crypt-villus axis, where it localizes to the apical surface. Closer inspection with super-resolution microscopy revealed that MISP exhibits strikingly specific enrichment on core bundle rootlets. In cultured cells, we found that MISP stabilizes and elongates rootlets, and recruits other canonical actin bundlers to these sites. Importantly, we found that purified MISP is sufficient to organize actin filaments into tight linear bundles in vitro. Finally, our preliminary analysis of MISP knockout mice revealed a striking loss of rootlets and decrease in microvillar surface density. Based on our preliminary data, we propose the following CENTRAL HYPOTHESIS: At the apical surface of differentiating enterocytes, MISP organizes actin filaments generated by the IRTKS/EPS8 complex to form core bundles that support the protrusion of brush border microvilli. Using a unique combination of state-of-the-art light and electron microscopy technology and novel biological model systems, we will: (Aim 1) determine if MISP specifies sites of microvillar growth at the apical surface, (Aim 2) define the mechanism of MISP actin binding and bundling, (Aim 3) elucidate the function of MISP in enterocyte differentiation in vivo. We expect that completion of these Aims will lead to new paradigms for understanding intestinal epithelial morphogenesis.

NIH Research Projects · FY 2025 · 2016-09

Project Summary: The Vanderbilt-Ingram Cancer Center (VICC) is an NCI-designated Comprehensive Cancer Center with over 270 members focused on cancer-related research. The Vanderbilt Institute of Chemical Biology (VICB) High- Throughput Screening (HTS) core facility was established to provide screening-based services for investigators for molecular probe and pharmacological discovery. Dr. Joshua Bauer plays an integral role in the interface between the cancer research and chemical biology programs at Vanderbilt. He has a strong background in cancer biology, pharmacology, and biochemistry. His main goal is to use chemical genomic approaches, including functional genomics screening and high-content imaging, to identify novel drug targets and to better understand the molecular and genetic mechanisms that underlie how cancer cells respond to therapeutics. To achieve this goal his cancer-related projects within the HTS facility are focused on three areas: 1) compound/drug library screening (i.e., HTS), 2) functional genomics (siRNA/CRISPR) library screening, and 3) high-content screening (HCS) and analysis. To date, Dr. Bauer has provided support and intellectual contributions to over 80 HTS projects, including collaborations with at least 41 Cancer Center members, including 31 that are NCI funded. These continued collaborations have allowed him to develop innovative assays and novel screening projects that contribute to the success of the Vanderbilt HTS core and cancer research program. His vital role and success stems from his ability to conceive, collaborate, design and perform screens, and intellectually contribute to projects, grant proposals, and manuscripts. In addition, through participation in conferences, meetings and workshops Dr. Bauer strives to bring state-of-the-art technologies and ideas to the Vanderbilt HTS facility. The blend of his skills, expertise, and knowledge provide a unique role within Vanderbilt to mend cancer research, chemical biology, genomics, drug discovery, and translational drug investigation. Through his previously funded R50 award, Dr. Bauer has already further advanced his areas of focus in HTS and his career development. So far on the award (2016-20), he has been co-authored on 11 papers, has supported 38 cancer-related grant proposals, supported and trained over 50 graduate students and postdocs on HTS instrumentation or software, has developed several novel functional genomics screens (CRISPR and ORFeome), and has pioneered several high-throughput 3D organoid models for high-content drug screening. Dr. Bauer has become a member of the NCI- Chemical Biology Consortium where he has been exposed to world-class drug discovery experts. Finally, the results of Dr. Bauer's work and collaborations have influenced the design of clinical trials, initiated sponsored research, and has led to lead molecules for further development. Therefore, Dr. Bauer's continued role as a Cancer Pharmacologist & HTS Scientist is completely indispensable to the cancer research program at Vanderbilt.

NIH Research Projects · FY 2026 · 2016-09

Project Summary The timely delivery of membrane-bound vesicles and tubules bearing transmembrane protein and lipid cargoes to discrete cellular membranes is fundamental to cell biology and human health. Many proteins associated with trafficking pathways are linked to serious and crippling human diseases, especially neurological diseases and disorders. Although many trafficking proteins and some pathways are well characterized, we still do not understand other trafficking pathways that we infer must exist between membranes. This constitutes an enormous gap in our understanding of fundamental cell biology. Our goal is to elucidate the molecular structures and functions of important coat protein complexes that initiate trafficking pathways by forming coats around vesicles or tubules. Coat proteins recognize and package relevant cargoes, and they promote efficient assembly of additional required protein components, like accessory proteins and SNAREs. Clathrin coats have long served as an important paradigm, but increasing evidence demonstrates how other coats use distinct mechanisms. We investigate the retromer and Assembly Polypeptide (AP) family of coat complexes (COPI, AP-4, AP-5) by using a variety of tools to ascertain molecular mechanisms of coat assembly and regulation. Biochemical approaches allow us to identify and test new interactions in coat complexes, including how accessory and regulatory proteins drive function. Integrated structural methods including X-ray crystallography, cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) provide detailed evidence for how coats interact with key partners and allow us to generate specific hypotheses to test function. Biophysical techniques enable us to quantify binding affinities and to probe interfaces identified in structural models. With collaborators, we use molecular data to design experiments in cultured cell lines or in model organisms to explore how protein-protein interactions drive phenotypes at the cellular or organismal levels. Ultimately, we hope to gain a molecular understanding of how coats assemble at distinct membranes to drive different trafficking pathways. We anticipate this work will reveal new mechanisms of coat assembly and regulation and will provide fundamental insights into the protein networks that underlie key cellular events on membranes.